>To: methods-and-reagents at net.bio.net>Subject: <none>
>Message-ID: <1995Jul13.192106.8868 at alder.cc.kcl.ac.uk>
>From: <rcha244 at bay.cc.kcl.ac.uk>
>Date: 13 Jul 95 19:21:06 GMT
>Nntp-Posting-Host: pc12.med.smd.kcl.ac.uk
>>Dear All -this is my first time so be gentle with me. I am enquiring
> on behalf of a colleague who requires an efficient, robust method
>for separating T and B lymphocytes.All suggestions welcome. I told my
> colleague the internet could do it. Hope I wont be disappointed.
>>Paul Crossey
>Dept of Medicine
>KCSMD
>London
>>email:rcha244 at bay.cc.kcl.ac.uk
Hi, Paul.
There was only one problem, there was left blank "Subject", what makes it
difficult to see what do you need, without "opening" the message.
Then, what is the source of your mixed population of T and B (is it blood)?
Then, if it is blood, what about macrophages (do they should be separated
from B cells)?
Then what kind of cells you are going deal with T or B or both?
Details, men, details!!!
Generally, you may choose two approaches (given for human heparinized blood).
1. Rosetting of T cells with AET-SRBC;
2. Centrifugation on Percol gradient.
The blood first applied on Ficol/hypaque (lymphoprep) allow separate PBMC.
In the former case, macrophages and B cells allowed to adhere on a surface
of culture dish, nonadherent,- T cells then mixed with AET treated SRBC.
The Rosettes thus formed, applied on Ficol/hypaque (lymphoprep). Due to
anchorage with SRBC only CD2+ (?) T cells will dive through gradient.
Subsequent lysis
of SRBC will leave up to 97% pure resting T cells.
In the second approach, PBMC applied on preformed Percol gradient, thus
separating macrophages from T and B. T cells could be separated from B by
allow B cells to adhere either on plastic, or glass beads, or nylon woolf,
what is better,- giving acceptable pure mixture of T cells.
What to choose depends from what you need. Rosetting gives you more
homogenous population of resting T cells. But Percol and nylon woolf
are less complicated to work with.
Hope this some of help.
Nikolai
http://128.252.119.253